Bottom Line:
These signaling networks specify the requirements of early and late events of peroxisome biogenesis.Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation.Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.

ABSTRACTReversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.

fig3: Negative effectors. (A) Oleate negative effectors. Pot1p-GFP levels in deletion mutants that exhibit an increase in Pot1p-GFP after 3 h of incubation in oleate are shown. At 6 h, the increase in Pot1p-GFP levels are generally higher than the population mean but show increased variability (right). (B) Glycerol negative effectors. The increase in Pot1p-GFP detected in deletion strains after 6 h of incubation in glycerol-containing medium is shown. Only the hsl1Δ mutant shows an up-regulation of Pot1p-GFP in both oleate and glycerol media, indicating that the normal role for HSL1 is as a negative effector in both carbon sources. The heat map shows the relative intensities of Pot1p-GFP fluorescence in these strains under the four media conditions tested, with red and green indicating an increase and a decrease in fluorescence, respectively. Error bars show SD.

Mentions:
We also identified a group of negative regulators whose deletion caused an increase in the expression of Pot1p-GFP in oleate (Fig. 3 A). This group (ark1Δ, hxk2Δ, kin3Δ, cdc19Δ, cla4Δ, hsl1Δ, and cln3Δ) does not include those mutants that show increased levels of Pot1p-GFP in glucose (Fig. 1 A) but rather those deletion strains that show normal glucose-repressed levels of Pot1p-GFP before the transition to an oleate-induced state (Fig. 3 A). In line with their roles as negative regulators, the population variability of these deletion strains is high (Fig. 3 A, right). Likewise, a set of deletion strains exhibited increased Pot1p-GFP fluorescence (>1 SD above wild-type levels) upon transition to glycerol (Fig. 3 B). It is noteworthy that only HSL1 negatively regulates both processes, again suggesting common stepwise elements to the derepression and activation of POT1 in the context of coordination with other cellular processes.

fig3: Negative effectors. (A) Oleate negative effectors. Pot1p-GFP levels in deletion mutants that exhibit an increase in Pot1p-GFP after 3 h of incubation in oleate are shown. At 6 h, the increase in Pot1p-GFP levels are generally higher than the population mean but show increased variability (right). (B) Glycerol negative effectors. The increase in Pot1p-GFP detected in deletion strains after 6 h of incubation in glycerol-containing medium is shown. Only the hsl1Δ mutant shows an up-regulation of Pot1p-GFP in both oleate and glycerol media, indicating that the normal role for HSL1 is as a negative effector in both carbon sources. The heat map shows the relative intensities of Pot1p-GFP fluorescence in these strains under the four media conditions tested, with red and green indicating an increase and a decrease in fluorescence, respectively. Error bars show SD.

Mentions:
We also identified a group of negative regulators whose deletion caused an increase in the expression of Pot1p-GFP in oleate (Fig. 3 A). This group (ark1Δ, hxk2Δ, kin3Δ, cdc19Δ, cla4Δ, hsl1Δ, and cln3Δ) does not include those mutants that show increased levels of Pot1p-GFP in glucose (Fig. 1 A) but rather those deletion strains that show normal glucose-repressed levels of Pot1p-GFP before the transition to an oleate-induced state (Fig. 3 A). In line with their roles as negative regulators, the population variability of these deletion strains is high (Fig. 3 A, right). Likewise, a set of deletion strains exhibited increased Pot1p-GFP fluorescence (>1 SD above wild-type levels) upon transition to glycerol (Fig. 3 B). It is noteworthy that only HSL1 negatively regulates both processes, again suggesting common stepwise elements to the derepression and activation of POT1 in the context of coordination with other cellular processes.

Bottom Line:
These signaling networks specify the requirements of early and late events of peroxisome biogenesis.Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation.Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.

ABSTRACTReversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.